scholarly journals MiR-181a Targets RSPO2 and Regulates Bone Morphogenetic Protein – WNT Signaling Crosstalk During Chondrogenic Differentiation of Mesenchymal Stromal Cells

2021 ◽  
Vol 9 ◽  
Author(s):  
Svitlana Melnik ◽  
Nina Hofmann ◽  
Jessica Gabler ◽  
Nicole Hecht ◽  
Wiltrud Richter
Keyword(s):  
Wnt Signaling ◽  
Bone Morphogenetic Protein ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Bmp Signaling ◽  
Cartilage Tissue ◽  
Signaling Crosstalk ◽  
Differentiation Potential ◽  
Morphogenetic Protein

Mechanisms of WNT and bone morphogenetic protein (BMP) signaling crosstalk is in the focus of multiple biological studies, and it also has been discovered to play important roles in human mesenchymal stromal cells (MSC) that are of great interest for neocartilage engineering due to their high chondrogenic differentiation potential. However, MSC-derived chondrocytes undergo hypertrophic degeneration that impedes their clinical application for cartilage regeneration. In our previous study, we established that several microRNAs (miRs) are differentially expressed between articular chondrocytes (AC) – and MSC-derived neocartilage, with miR-181a being the most prominent candidate as key microRNA involved in the regulation of a balance between chondral and endochondral differentiation. The aim of this study was the identification of precise mRNA targets and signaling pathways regulated by miR-181a in MSC during chondrogenesis. MiR-181a was upregulated during chondrogenesis of MSC, along with an increase of the hypertrophic phenotype in resulting cartilaginous tissue. By in silico analysis combined with miR reporter assay, the WNT signaling activator and BMP signaling repressor RSPO2 was suggested as a target of miR-181a. Further validation experiments confirmed that miR-181a targets RSPO2 mRNA in MSC. It was found that in human MSC miR-181a activated BMP signaling manifested by the accumulation of SOX9 protein and increased phosphorylation of SMAD1/5/9. These effects, together with the concomitant reduction of canonical WNT signaling induced by miR-181a mimic, were in accordance with the effects expected by the loss of RSPO2, thus indicating the causative link between miR-181a and RSPO2. Moreover, we observed that a tight correlation between miR-181a and miR-218 expression levels in healthy human cartilage tissue was disrupted in osteoarthritis (OA) highlighting the importance of the WNT-BMP signaling crosstalk for preventing OA.

scholarly journals MiR-218 affects hypertrophic differentiation of human mesenchymal stromal cells during chondrogenesis via targeting RUNX2, MEF2C, and COL10A1

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Svitlana Melnik ◽  
Jessica Gabler ◽  
Simon I. Dreher ◽  
Nicole Hecht ◽  
Nina Hofmann ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Expression Profile ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Cartilage Tissue ◽  
Protein Accumulation ◽  
Differentiation Potential ◽  
Human Mesenchymal Stromal Cells ◽  
Alp Activity ◽  
Pulldown Assay

Abstract Background Human mesenchymal stromal cells (MSC) hold hopes for cartilage regenerative therapy due to their chondrogenic differentiation potential. However, undesirable occurrence of calcification after ectopic transplantation, known as hypertrophic degeneration, remains the major obstacle limiting application of MSC in cartilage tissue regeneration approaches. There is growing evidence that microRNAs (miRs) play essential roles in post-transcriptional regulation of hypertrophic differentiation during chondrogenesis. Aim of the study was to identify new miR candidates involved in repression of hypertrophy-related targets. Methods The miR expression profile in human articular chondrocytes (AC) was compared to that in hypertrophic chondrocytes derived from human MSC by microarray analysis, and miR expression was validated by qPCR. Putative targets were searched by in silico analysis and validated by miR reporter assay in HEK293T, by functional assays (western blotting and ALP-activity) in transiently transfected SaOS-2 cells, and by a miR pulldown assay in human MSC. The expression profile of miR-218 was assessed by qPCR during in vitro chondrogenesis of MSC and re-differentiation of AC. MSC were transfected with miR-218 mimic, and differentiation outcome was assessed over 28 days. MiR-218 expression was quantified in healthy and osteoarthritic cartilage of patients. Results Within the top 15 miRs differentially expressed between chondral AC versus endochondral MSC differentiation, miR-218 was selected as a candidate miR predicted to target hypertrophy-related genes. MiR-218 was downregulated during chondrogenesis of MSC and showed a negative correlation to hypertrophic markers, such as COL10A1 and MEF2C. It was confirmed in SaOS-2 cells that miR-218 directly targets hypertrophy-related COL10A1, MEF2C, and RUNX2, as a gain of ectopic miR-218 mimic caused drop in MEF2C and RUNX2 protein accumulation, with attenuation of COL10A1 expression and significant concomitant reduction of ALP activity. A miR pulldown assay confirmed that miR-218 directly targets RUNX2, MEF2C in human MSC. Additionally, the gain of miR-218 in human MSC attenuated hypertrophic markers (MEF2C, RUNX2, COL10A1, ALPL), although with no boost of chondrogenic markers (GAG deposition, COL2A1) due to activation of WNT/β-catenin signaling. Moreover, no correlation between miR-218 expression and a pathologic phenotype in the cartilage of osteoarthritis (OA) patients was found. Conclusions Although miR-218 was shown to target pro-hypertrophic markers MEF2C, COL10A1, and RUNX2 in human MSC during chondrogenic differentiation, overall, it could not significantly reduce the hypertrophic phenotype or boost chondrogenesis. This could be explained by a concomitant activation of WNT/β-catenin signaling counteracting the anti-hypertrophic effects of miR-218. Therefore, to achieve a full inhibition of the endochondral pathway, a whole class of anti-hypertrophic miRs, including miR-218, needs to be taken into consideration.

scholarly journals Amniotic Mesenchymal Stromal Cells Exhibit Preferential Osteogenic and Chondrogenic Differentiation and Enhanced Matrix Production Compared With Adipose Mesenchymal Stromal Cells

2017 ◽  
Vol 45 (11) ◽  
pp. 2637-2646 ◽  
Author(s):  
Natasha Topoluk ◽  
Richard Hawkins ◽  
John Tokish ◽  
Jeremy Mercuri
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Cell Types ◽  
Cell Counting ◽  
Gene Transcript ◽  
Differentiation Potential ◽  
Musculoskeletal Tissue ◽  
Cell Groups ◽  
And Cartilage

Background: Therapeutic efficacy of various mesenchymal stromal cell (MSC) types for orthopaedic applications is currently being investigated. While the concept of MSC therapy is well grounded in the basic science of healing and regeneration, little is known about individual MSC populations in terms of their propensity to promote the repair and/or regeneration of specific musculoskeletal tissues. Two promising MSC sources, adipose and amnion, have each demonstrated differentiation and extracellular matrix (ECM) production in the setting of musculoskeletal tissue regeneration. However, no study to date has directly compared the differentiation potential of these 2 MSC populations. Purpose: To compare the ability of human adipose- and amnion-derived MSCs to undergo osteogenic and chondrogenic differentiation. Study Design: Controlled laboratory study. Methods: MSC populations from the human term amnion were quantified and characterized via cell counting, histologic assessment, and flow cytometry. Differentiation of these cells in comparison to commercially purchased human adipose-derived mesenchymal stromal cells (hADSCs) in the presence and absence of differentiation media was evaluated via reverse transcription polymerase chain reaction (PCR) for bone and cartilage gene transcript markers and histology/immunohistochemistry to examine ECM production. Analysis of variance and paired t tests were performed to compare results across all cell groups investigated. Results: The authors confirmed that the human term amnion contains 2 primary cell types demonstrating MSC characteristics—(1) human amniotic epithelial cells (hAECs) and (2) human amniotic mesenchymal stromal cells (hAMSCs)—and each exhibited more than 90% staining for MSC surface markers (CD90, CD105, CD73). Average viable hAEC and hAMSC yields at harvest were 2.3 × 106 ± 3.7 × 105 and 1.6 × 106 ± 4.7 × 105 per milliliter of amnion, respectively. As well, hAECs and hAMSCs demonstrated significantly greater osteocalcin ( P = .025), aggrecan ( P < .0001), and collagen type 2 ( P = .044) gene expression compared with hADSCs, respectively, after culture in differentiation medium. Moreover, both hAECs and hAMSCs produced significantly greater quantities of mineralized ( P < .0001) and cartilaginous ( P = .0004) matrix at earlier time points compared with hADSCs when cultured under identical osteogenic and chondrogenic differentiation conditions, respectively. Conclusion: Amnion-derived MSCs demonstrate a greater differentiation potential toward bone and cartilage compared with hADSCs. Clinical Relevance: Amniotic MSCs may be the source of choice in the regenerative treatment of bone or osteochondral musculoskeletal disease. They show significantly higher yields and better differentiation toward these tissues than MSCs derived from adipose.

scholarly journals Loss of mismatch repair signaling impairs the WNT–bone morphogenetic protein crosstalk and the colonic homeostasis

2019 ◽  
Vol 12 (6) ◽  
pp. 410-423
Author(s):  
Katrine Nørgaard ◽  
Carolin Müller ◽  
Nadja Christensen ◽  
María L Chiloeches ◽  
Cesilie L Madsen ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Mismatch Repair ◽  
Goblet Cells ◽  
Bmp Signaling ◽  
Colonic Epithelium ◽  
Signaling Crosstalk ◽  
Dna Mismatch ◽  
Morphogenetic Protein ◽  
And Function ◽  
Deficient Mice

Abstract The fine balance between proliferation, differentiation, and apoptosis in the colonic epithelium is tightly controlled by the interplay between WNT, Notch, and bone morphogenetic protein (BMP) signaling. How these complex networks coordinate the colonic homeostasis, especially if cancer predisposing mutations such as mutations in the DNA mismatch repair (MMR) are present, is unclear. Inactivation of the MMR system has long been linked to colorectal cancer; however, little is known about its role in the regulation of the colonic homeostasis. It has been shown that loss of MMR promotes the proliferation of colon epithelial cells that renders them highly susceptible to transformation. The mechanism through which MMR mediates this effect, yet, remains to be determined. Using an MMR-deficient mouse model, we show that increased methylation of Dickkopf1 impacts its expression, and consequently, the ability to negatively regulate WNT signaling. As a result, excessive levels of active β-catenin promote strong crypt progenitor-like phenotype and abnormal proliferation. Under these settings, the development and function of the goblet cells are affected. MMR-deficient mice have fewer goblet cells with enlarged mucin-loaded vesicles. We further show that MMR inactivation impacts the WNT–BMP signaling crosstalk.

scholarly journals Genetic Markers Can Predict Chondrogenic Differentiation Potential in Bone Marrow-Derived Mesenchymal Stromal Cells

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Masami Kanawa ◽  
Akira Igarashi ◽  
Katsumi Fujimoto ◽  
Yukihito Higashi ◽  
Hidemi Kurihara ◽  
...  
Keyword(s):  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Chondrogenic Differentiation ◽  
Expression Profiles ◽  
Three Dimensional ◽  
Marker Genes ◽  
Differentiation Potential ◽  
Expression Levels ◽  
Group 2 ◽  
Msc Differentiation

The precise predictions of the differentiation direction and potential of mesenchymal stromal cells (MSCs) are an important key to the success of regenerative medicine. The expression levels of fate-determining genes may provide tools for predicting differentiation potential. The expression levels of 95 candidate marker genes and glycosaminoglycan (GAG) contents after chondrogenic induction in 10 undifferentiated ilium and 5 jaw MSC cultures were determined, and their correlations were analyzed. The expression levels of eight genes before the induction of chondrogenic MSC differentiation were significantly correlated with the GAG levels after induction. Based on correlation patterns, the eight genes were classified into two groups: group 1 genes (AURKB, E2F1, CDKN2D, LIF, and ACLY), related to cell cycle regulation, and group 2 genes (CD74, EFEMP1, and TGM2), involved in chondrogenesis. The expression levels of the group 2 genes were significantly correlated with the ages of the cell donors. The expression levels of CDKN2D, CD74, and TGM2 were >10-fold higher in highly potent MSCs (ilium MSCs) than in MSCs with limited potential (jaw MSCs). Three-dimensional (3D) scatter plot analyses of the expression levels of these genes showed reduced variability between donors and confirmed predictive potential. These data suggest that group 2 genes are involved in age-dependent decreases in the chondrogenic differentiation potential of MSCs, and combined 3D analyses of the expression profiles of three genes, including two group 2 genes, were predictive of MSC differentiation potential.

DEVELOPMENT OF OSTEOPLASTIC MATERIAL IMPREGNATED WITH PLASMID ENCODING BONE MORPHOGENETIC PROTEIN-2

2020 ◽  
pp. 63-65
Author(s):  
T. Bukharova ◽  
A. Vasilyev ◽  
A. Kulakov ◽  
D. Goldstein
Keyword(s):  
Bone Morphogenetic Protein ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Bone Morphogenetic Protein 2 ◽  
Morphogenetic Protein ◽  
New Gene ◽  
Osteoplastic Material

A new gene-activated osteoplastic based on chitosan material impregnated with plasmid with the BMP-2 gene has been developed. The material had an osteoinductive effect on mesenchymal stromal cells (MSC).

Development of osteoplastic Material Impregnated with Plasmid Encoding Bone Morphogenetic Protein-2

2020 ◽  
Vol 36 (4) ◽  
pp. 59-64
Author(s):  
T.B. Bukharova ◽  
Yu.D. Zagoskin ◽  
A.V. Vasilyev ◽  
T.E. Grigoriev ◽  
V.O. Mokrousova ◽  
...  
Keyword(s):  
Bone Morphogenetic Protein ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Multipotent Mesenchymal Stromal Cells ◽  
Biological Properties ◽  
Bone Morphogenetic Protein 2 ◽  
Russian Science ◽  
Morphogenetic Protein ◽  
Osteoplastic Material

The biological properties of a new gene-activated osteoplastic material based on chitosan sponges impregnated with plasmids with the gene for the bone morphogenetic protein-2 (BMP2) have been studied. The optimal conditions for transfection of multipotent mesenchymal stromal cells with TurboFect reagent were selected. The in vitro osteoinducing effect of the developed material associated with an increase in the expression of osteogenic markers in mesenchymal stromal cells was shown. The designed gene-activated material can be used to repair bone defects. gene-activated material, plasmids, BMP-2, gene therapy, bone regeneration The work was supported by the Russian Science Foundation (Grant no. 16-15-00298).

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